Bot. Bull. Acad. Sin. (2004) 45: 41-48

Hsu and Kao — Abscisic acid and phosphinothricin-tolerant rice seedlings

Phosphinothricin tolerance in rice (Oryza sativa L.) seedlings is associated with elevated abscisic acid in the leaves

Yi Ting Hsu and Ching Huei Kao*

Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China

(Received March 18, 2003; Accepted August 29, 2003)

Abstract. Phosphinothricin (PPT, known as glufosinate) is a nonselective herbicide that is a potent inhibitor of glutamine synthetase (GS). PPT-tolerant and PPT-sensitive rice cultivars were used to study the role of abscisic acid (ABA) in PPT tolerance. ABA content was determined by enzyme-linked immunosorbent assay. PPT toxicity was evaluated by the decrease in chlorophyll and protein contents in the leaves. On treatment with PPT, ABA content significantly increased in the leaves of the PPT-tolerant cultivars (cv. Tainung 67, TNG67) but not in the PPT-sensitive cultivar (cv. Taichung Native 1, TN1). The reduction of transpiration rate of TN1 seedlings caused by PPT was less than that of TNG67. Pretreatment with ABA enhanced PPT tolerance and reduced PPT-induced NH4+ accumulation in leaves of TN1. Exogenous application of the ABA biosynthesis inhibitor, fluridone, decreased PPT tolerance and increased NH4+ content in the leaves of TNG 67. Fluridone effect on PPT toxicity of TNG67 seedlings was reversed by the application of ABA. Evidence is presented to show that PPT-induced NH4+ accumulation, rather than depletion of glutamine, is the mechanism that regulates PPT-induced toxicity in TN1 leaves. In conclusion, the increase in endogenous ABA content in the leaves is closely related to PPT tolerance of rice seedlings.

Keywords: Abscisic acid; Ammonium ion toxicity; Glutamine synthetase; Phosphinothricin tolerance; Rice.

Abbreviations: ABA, abscisic acid; ELISA, enzyme-linked immunosorbent assay; f.wt, fresh weight; GS, glutamine synthetase; PPT, phosphinothricin; TN1, Taichung Native 1; TNG67, Tainung 67.

Introduction

Glutamine synthetase (GS, EC 6.3.1.2) is the initial enzyme in the pathway that assimilates inorganic nitrogen into organic compounds (Lea and Miflin, 1974). It is an important enzyme in nitrogen metabolism in that, in addition to assimilating NH4+ produced by nitrite reductase, reassimilates NH4+ released as a result of photorespiration and the breakdown of proteins and nitrogen transport compounds (Miflin and Habash, 2002). Phosphinothricin [PPT, 2-amino-4-(methylphosphinyl)-butanoic acid, known as glufossinate] is a nonselective herbicide that is a potent inhibitor of GS (Leason et al., 1982; Ridley and McNally, 1985). Inhibition of GS activity by PPT leads to a rapid accumulation of NH4+ under conditions in which nitrite is being photosynthetically reduced and/or conditions which support photorespiration (Tachinbana et al., 1986; Wild et al., 1987; Sauer et al., 1987; Wendler et al., 1990). A high level of NH4+ is known to have a toxic effect on plant cells (Givan, 1979). Thus, PPT toxicity may be a direct result of NH4+ accumulation.

The plant hormone abscisic acid (ABA) is a sesquiterpenoid synthesized from xanthophylls (Taylor et al., 2000; Seo and Koshiba, 2002) and appears to influence several physiological and developmental events (Zeevaart

and Creelman, 1988; Kende and Zeevaart, 1997). The level of ABA in plants increases upon their exposure to environmental stress (Zeevaart and Creelman, 1988; Xu et al., 1995). ABA content has been correlated with increased tolerance to freezing (Guy, 1990), chilling (Lee et al., 1993), drought (Zeevaart and Creelman, 1988), salt (LaRose et al., 1987), and cadmium toxicity (Hsu and Kao, 2003).

It has been shown that ABA accumulation is a common effect of using auxin herbicides on susceptible plants (Grossmann et al., 1996; Grossmann, 2000; Hansen and Grossmann, 2000). Recently, we demonstrated that PPT treatment resulted in an increase in ABA level in detached rice leaves (Tsai et al., 2002). However, no correlation between ABA and PPT toxicity or PPT tolerance in detached rice leaves could be established (Tsai et al., 2002). Our preliminary observation showed that rice seedlings of cultivar Tainung 67 (TNG67) are more tolerant to PPT than those of cultivar Taichung Native 1 (TN1) when PPT was added directly to the culture solution. It appears that these two cultivars of rice seedlings with different tolerance to PPT provide a useful system to study mechanism of PPT tolerance of rice plants. In this investigation, we examine the role of ABA in PPT tolerance of rice seedlings.

Materials and Methods

Plant Cultivation and Treatment

Two rice (Oryza sativa L.) cultivars, an Indica type cultivar TN1 and a Japonica cultivar, TNG67, obtained from

*Corresponding author. Tel: 886-2-23698159; Fax: 886-2-23620879; E-mail: kaoch@ntu.edu.tw


Botanical Bulletin of Academia Sinica, Vol. 45, 2004

Taiwan Agricultural Research Institute, Taichung, Taiwan, were used in this study. Seeds were sterilized with 2.5% sodium hypochlorite for 15 min and washed extensively with distilled water. These seeds were then germinated in a Petri dish on wetted filter papers at 37 °C in the dark. After 48 h incubation, uniformly germinated seeds were selected and cultivated in a 250 ml beaker containing half-strength Kimura B solution as described previously (Chu and Lee, 1989). The concentrations of N, P, K, S, Ca and Mg in half-strength Kimura B solution are 11.5, 2.9, 7.2, 15.0, 7.4 and 8.7 ppm, respectively. The hydroponically cultivated seedlings were grown in a Phytotron with natural light at 30 °C day (12 h)/25 °C night (12 h) and 90% relative humidity. Twelve_day-old seedlings with three leaves were used in all experiments.

For PPT, ABA, and fluridone treatments, the chemicals were added directly to the culture solution during the experiment.

Determinations of Chlorophyll, Protein, NH4+, and Glutamine

Chlorophyll was determined according to Wintermans and De Mots (1965) after extraction in 96% (v/v) ethanol. For protein extraction, leaf segments were homogenized in 50 mM sodium phosphate buffer (pH 6.8). The extracts were centrifuged at 17,600 g for 20 min, and the supernatants were used for determination of protein by the method of Bradford (1976). Ammonium ion was extracted by homogenizing leaf samples in 0.3 mM sulphuric acid (pH 3.5). The homogenate was centrifuged for 10 min at 39,000 g and the supernatant was used for determination of NH4+ as described by Lin and Kao (1996). For determination of glutamine, leaf samples were extracted with 2% sulfosalicylic acid, and the homogenates were centrifuged at 15,000 g for 20 min. The supernatant was used directly for amino acid analysis (amino acid analyzer, Beckmann 6300, Palo Alto, USA). All measurements are expressed on the basis of f. wt.

GS Activity

For extraction of GS, leaf segments were homogenized with 10 mM Tris-HCl buffer (PH 7.6, containing 1 mM MgCl2, 1 mM EDTA, and 1 mM 2-mercaptoethanol) using a chilled mortar and pestle. The homogenate was centrifuged at 15,000 g for 30 min, and the resulting supernatant was used for determination of GS activity. The whole extraction procedure was carried out at 4°C. GS was assayed by the method of Oaks et al. (1980). The reaction mixture contained in a final volume of 1 ml was 80 µmol Tris-HCl buffer, 40 µmol L-glutamic acid, 8 µmol ATP, 24 µmol MgSO4, and 16 µmol NH2OH; the final pH was 8.0. The reaction was started by addition of the enzyme extract and after incubation for 30 min at 30°C, it was stopped by adding 2 ml 2.5% (w/v) FeCl3 and 5% (w/v) trichloroacetic acid in 1.5 M HCl. After centrifugation the absorbance of the supernatant was read at 540 nm. One unit of GS activity was defined as 1 µmol L-glutamate g-monohydroxamate formed per min. GS activity is expressed on the basis of f. wt.

ABA Determination

For extraction of ABA, leaves were homogenized with a mortar and pestle in extraction solution (80% methanol containing 2% glacial acetic acid). To remove plant pigments and other non-polar compounds which could interfere in the immunoassay, extracts were first passed through polyvinylprrolidone column and C18 cartridges. The elutes were concentrated to dryness by vacuum-evaporation and resuspended in Tris-buffered saline before enzyme-linked immunosorbent assay (ELISA). ABA was quantified by ELISA (Walker-Simmons, 1987). ABA immunoassay detection kit (PGR-1) was purchased from Sigma Chemical Co. (St Louis, MO, USA) and is specific for (+)-ABA. By evaluating 3H-ABA recovery, ABA loss was less than 3% by the method described here. ABA content is expressed on the basis of f. wt.

Transpiration Rate

The transpiration rate was measured according to Greger and Johansson (1992). The transpiration rate was calculated from the water loss during each interval and converted to a per day, per seedlings basis.

Experimental Design

In our investigation, the seedlings were grown for 12 days in a greenhouse, where natural light was provided. The growth of rice seedlings is very sensitive to light and varies with different light intensities. Experiments were carried out at different times of the year. Thus, the absolute levels of each measurement varied among experiments because of seasonal effects. However, the patterns of response to PPT were reproducible. For all measurements, each treatment was performed four times. All experiments described here were performed three times. Similar results and identical trends were obtained each time. The data reported here are from a single experiment.

Results

Evaluation of PPT Tolerance

When rice seedlings were treated with 10 mM PPT for 7 days, leaf chlorosis was observed in TN1 seedlings, but not in TNG67 seedlings (Figure 1). In short term (3 days) experiments, chlorosis was first observed in the second leaf of TN1 seedlings. Thus, PPT toxicity in the second leaf was assessed by a decrease in chlorophyll and protein contents. Increasing concentration of PTT from 3 to 10 µM progressively decreased chlorophyll and protein contents in leaves of TN1 seedlings (Figure 2). However, the decrease in chlorophyll and protein contents in leaves of TNG67 seedlings was not observed (Figure 2). PPT at 10 µM seems to be the optimum concentration in inducing toxicity. Figure 3 also shows PPT (10 µM) had no effect on chlorophyll and protein contents in leaves of TNG67 seedlings, but significantly decreased chlorophyll and protein contents in leaves of TN1 seedlings. All these results suggest that TNG67 seedlings are apparently more tolerant to PPT than TN1 seedlings.


Hsu and Kao — Abscisic acid and phosphinothricin-tolerant rice seedlings

Ammonium Ion Content and GS Activity

Under PPT treatment NH4+ accumulated significantly in leaves of TN1 seedlings (Figure 3). However, none accumulated in the second leaf of TNG67 seedlings treated with PPT. GS is the primary enzyme responsible for NH4+ assimilation in plants (Lea and Miflin, 1974; Miflin and Habash, 2002). We observed that GS activity in the second leaf of TN1 treated with PPT decreased compared with that given no PPT treatment (Figure 3). In contrast, PPT had no effect on the GS activity of the second leaf of TNG67 seedlings (Figure 3).

The decrease in GS activity by PPT in the second leaf possibly results from the direct effect of PPT. This possibility was tested by mixing enzyme extract of the second leaf with various concentrations of PPT. GS activity was then assayed 30 min after mixing. It was found that GS activity was directly inhibited by PPT in both TNG67 and TN1 leaf extracts (Figure 4). Thus, the lack of inhibition of GS activity in the second leaf of TNG67 seedlings is mainly due to the lack of increase in PPT content. Current results also suggest that PPT-induced NH4+ accumulation in the second leaf of TN1 seedlings is attributable to the decrease in GS activity.

Transpiration Rate

The transpiration rate of TN1 seedlings was observed to be higher than that of TNG67 seedlings (Table 1). PPT treatment decreased this rate in both TN1 and TNG 67 seedlings (Table 1). However, the decrease was less pronounced in the former (Table 1).

Figure 1. Effect of phosphinothricin (PPT, 10 µM) on leaf chlorosis of Taichung Native 1 (TN1) and Tainung 67 (TNG67) rice seedlings. Picture was taken after 7 days of treatment.

Figure 2. Effect of phosphinothricin (PPT, 10 µM) concentrations on chlorophyll and protein contents in the second leaf rice seedlings. Chlorophyll and protein contents were measured after 2 days of treatments. Data are means (± SE) of four replicates of a single typical experiment. Three series of independent experiments were carried out giving reproducible results.

Figure 3. Effect of phosphinothricin (PPT, 10 µM) on the contents of chlorophyll, protein, NH4+ and the activity of glutamine synthetase (GS) in the second leaf of Taichung Native 1 (TN1) and Tainung 67 (TNG67) rice seedlings. All measurements were made after 2 days of treatment. Data are means (± SE) of four replicates of a single typical experiment. Three series of independent experiments were carried out giving reproducible results.


Botanical Bulletin of Academia Sinica, Vol. 45, 2004

ABA pretreatment resulted in no further enhancement of PPT tolerance in TNG67 leaves (Figure 7), indicating that the amount of ABA in PPT-treated TNG67 leaves is sufficient to exert its effect on PPT tolerance. As expected, ABA pretreatment reduced PPT-induced NH4+ accumulation and PPT-decreased GS activity in the third leaf of TN1 seedlings (Figure 8).

Fluridone Treatment

The role of ABA in PPT tolerance was further tested using an inhibitor of ABA biosynthesis fluridone, which blocks the conversion of phytoene to phytofluene in carotenoid biosynthesis pathway (Kowalczyk-Schröder and Sandmann, 1992). Fluridone was observed to inhibit the

Figure 4. Effect of phosphinothricin (PPT) on the activity of GS extracted from rice leaves. Extracts were prepared from the second leaf of 12-day-old Taichung Native 1 (TN1) and Tainung 67 (TNG67) rice seedlings. Assays were carried out 30 min after the addition of various concentrations of PPT. Each treatment was performed four times. The data reported here were from one of three independent experiments.

Glutamine Content

An inhibition of GS activity in the second leaf of TN1 seedlings may also result in a decrease in glutamine. Depletion of glutamine could be another reason for PPT-induced toxicity of TN1 seedlings. To test this possibility, we measured the content of glutamine in the second leaf of rice seedlings either untreated or treated with PPT. Contrary to our expectation, PPT-treated second leaf of TN1 seedlings had more glutamine than control leaves (Figure 5). Higher glutamine content in the second leaf of TNG67 seedlings was observed only at 3 days after PPT treatment (Figure 5).

ABA Content

Changes in endogenous ABA in leaves of seedlings treated with or without PPT are shown in Figure 6. In TN1 seedlings, no differences in ABA content were observed between PPT-treated leaves and controls. However, ABA content in PPT-treated TNG67 leaves was observed to be higher than in controls after 2 days of treatment. It is evident that ABA accumulates greater in TNG67 seedlings than in TN1 seedlings in response to PPT.

Pretreatment with ABA

If ABA plays an important role in PPT tolerance, then pretreatment of TN1 seedlings with ABA could be expected to reduce PPT toxicity, PPT-induced NH4+ accumulation, and PPT-decreased GS activity in TN1 leaves. Since some chlorosis was observed in the second leaf of TN1 treated with ABA (5 µM) for 2 days, PPT toxicity was evaluated using the third leaf. ABA pretreatment reduced PPT toxicity in the third leaf of TN1 seedlings (Figure 7). However,

Figure 5. Changes in glutamine content in the second leaf of Taichung Native 1 (TN1) and Tainung 67 (TNG67) rice seedlings either untreated or treated with phosphinothricin (PPT, 10 mM). Data are means (± SE) of four replicates of a single typical experiment. Three series of independent experiments were carried out giving reproducible results.

Figure 6. Changes in ABA content in the second leaf of Taichung Native 1 (TN1) and Tainung 67 (TNG67) rice seedlings either untreated or treated with phosphinothricin (PPT, 10 mM). Data are means (± SE) of four replicates of a single typical experiment. Three series of independent experiments were carried out giving reproducible results.


Hsu and Kao — Abscisic acid and phosphinothricin-tolerant rice seedlings

Figure 7. Effect of ABA-pretreatment on the contents of chlorophyll and protein in the third leaf of Taichung Native 1 (TN1) and Tainung 67 (TNG67) rice seedlings either untreated or treated with phosphinothricin (PPT, 10 mM). Rice seedlings were pretreated with ABA (5 mM) for 2 days and then either untreated or treated with PPT for 2 days. Data are means (± SE) of four replicates of a single typical experiment. Three series of independent experiments were carried out giving reproducible results.

increase in ABA content (Table 2) and enhance PPT toxicity (Figure 9), NH4+ accumulation, and the decrease in GS activity (Figure 10) in leaves of TNG67. Figure 9 also shows that the effect of fluridone on PPT toxicity in leaves of TNG67 seedlings can be rescued by the application of 2 µM ABA. These results further strengthened the role of ABA on PPT tolerance of rice seedlings. Fluridone treatment, however, did not enhance PPT toxicity in leaves of TN1 seedlings (Figure 9).

Discussion

Our study indicates that ABA is involved in the PPT tolerance of rice seedlings. This conclusion was based on the observation that: (a) ABA accumulated more in TNG67 seedlings, a PPT tolerant cultivar, than in TN1 seedlings, a PPT sensitive cultivar, in response to PPT (Figure 6); (b) exogenous application of ABA increased the PPT tolerance of TN1 seedlings (Figure 7); (c) fluridone treatment reduced ABA content (Table 2), as well as PPT tolerance of TNG67 seedlings (Figure 9); (d) the effect of fluridone on the PPT toxicity of TNG67 seedlings can be reversed by the application of ABA (Figure 9). The results suggest that the regulation of endogenous ABA biosynthesis by PPT, applied directly to culture solution, is causally correlated to the PPT tolerance of rice seedlings. Since fluridone is an inhibitor of ABA biosynthesis through carotenoid pathway (Kowalczyk-Schröder and Sandmann, 1992), the effects of this inhibitor on TNG67

Figure 8. Effect of ABA-pretreatment on the content of NH4+ and the activity of GS in the third leaf of Taichung Native 1 rice seedlings either untreated or treated with phosphinothricin (PPT, 10 mM). Rice seedlings were pretreated or treated with ABA (5 mM) for 2 days and then either untreated or treated with PPT for 2 days. Data are means (± SE) of four replicates of a single typical experiment. Three series of independent experiments were carried out giving reproducible results.


Botanical Bulletin of Academia Sinica, Vol. 45, 2004

may imply that the ABA biosynthetic pathway in response to PPT appears to be the same as that established under stress conditions (Zeevaart and Creelman, 1988; Seo and Koshiba, 2002). In addition, the defect of ABA accumulation in TN1 seedlings may account for the PPT intolerance of the cultivar.

In previous work, we observed that PPT at a concentration of 50 µM increased ABA content in detached rice leaves of TN1 (Tsai et al., 2002). In the present investigation, no accumulation of ABA was observed in leaves of TN1 seedlings treated with 10 µM PPT (Figure 6). When detached rice leaves of TN1 were treated with 10 µM PPT, no ABA accumulation was observed (data not shown). It appears that PPT concentration in leaves of TN1 seedlings treated with 10 µM PPT is high enough to cause toxicity, but not to cause ABA accumulation. It has been shown that roots possess the ability to synthesize ABA (Davies and Zhang, 1991). Thus, the possibility that PPT is unable to trigger the ABA biosynthetic pathway in TN1 roots cannot be excluded.

It has been shown that ABA accumulation is a common effect of auxin herbicides in susceptible plants (Grossmann et al., 1996; Grossmann, 2000; Hansen and Grossmann, 2000). PPT, a GS inhibiter, is widely used as a nonselective herbicide. Here, we demonstrate, for the first time, that ABA accumulation was observed only in PPT tolerant rice seedlings.

It is generally considered that inhibition of GS by PPT leads to the accumulation of NH4+ and depletion of glutamine in treated tissue (Lea et al., 1984; Hurst et al., 1993; Downs et al., 1994). PPT-induced NH4+ accumulation appears to be the mechanism that regulates PPT-induced toxicity in TN1 leaves. This conclusion is based on the observations that: (a) PPT toxicity is associated with the increased NH4+ level in TN1 leaves (Figure 3), (b) exogenous application of ABA decreased PPT-induced NH4+ accumulation and PPT-induced toxicity as well in TN1 leaves (Figures 7 and 8), and (c) PPT toxicity cannot be explained by the depletion of glutamine content in TN1 leaves (Figure 5).

TNG67 seedlings have significantly smaller leaves and less stomata per unit leaf area than TN1 seedlings (Figure 1, Lin et al., 2001), which may lead to lower transpiration rates in TNG67 leaves (Table 1). We also observed that PPT decreased the transpiration rate of both cultivars (Table 1). PPT treatment reduced transpiration rate in TN1 and TNG67 to about 48% and 85% of the control value, respectively (Table 1). Thus, the reduction of transpiration rate of TN1 seedlings caused by PPT, which are unable to accumulate ABA, was less than that of TNG67 seedlings, which accumulate ABA, and consequently may have a higher PPT content. In this study, we are unable to provide direct evidence that TNG67 leaves have less PPT than TN1 leaves. However, the facts that PPT directly inhibits GS activity extracted from TNG67 leaves (Figure 4) and that PPT fails to inhibit GS activity in the second leaf of TNG67 (Figure 3) provide circumstantial evidence that TNG67 leaves may have a negligible amount of PPT.

Figure 9. Effect of fluridone (Flu, 0.2 mM) or the content of chlorophyll and protein contents in the second leaf of Taichung Native 1 (TN1) and Tainung 67 (TNG67) rice seedlings either untreated or treated with phosphinothricin (PPT, 10 mM) or ABA (2 mM). Chlorophyll and protein contents were measured after 2 days of treatment. Data are means (± SE) of four replicates of single typical experiment. Three series of independent experiments were carried out giving reproducible results.

Figure 10. Effect of fluridone (Flu, 0.2 mM) on the content of NH4+ and the activity of GS in the second leaf of Tainung 67 rice seedlings either untreated or treated with phosphinothricin (PPT, 10 mM). All measurements were made after 2 days of treatment. Data are means (± SE) of four replicates of single typical experiment. Three series of independent experiments were carried out giving reproducible results.


Hsu and Kao — Abscisic acid and phosphinothricin-tolerant rice seedlings

It appears that an increase in endogenous ABA content is closely related to the PPT tolerance of rice seedlings. ABA may exert its regulatory effect on transpiration rate, which reduces the translocation of PPT to the shoot.

There are reports that plants are able to reduce the toxic effect of the PPT by metabolizing PPT to stable and inactive compounds like 4-methylphosphinico-2-hydroxy-butanoic acid and its derivatives by non-specific actylation reactions (Dröge-Laser et al., 1994; Jansen and Schimidt, 2000; Müller et al., 2001). It is not known whether ABA is effective in metabolizing PPT in TNG67 leaves. This would be an interesting direction for our future study.

Acknowledgments. This work was financially supported by Grant NSC 91-2313-B002-364 from the National Science Council of the Republic of China.

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